It is key to any analysis of a biological sample that the integrity of its constituents is conserved between the time that the sample is extracted from a living organism and the time that analysis is carried out. Sample degradation, however, is both hard to impede, and hard to detect. The result is that many analyses miss the presence of species that have degraded long before the analysis is carried out; correspondingly, such analyses may in fact identify degradation products of critical components in place of the original components.
Since the sequencing of the human genome and the realization that there may be far fewer genes than was originally thought, attention has turned to the proteome; it is now believed that it is the assemblage of proteins in an organism that is the key to understanding physiology, disease, and function. Proteins are found in many different environments, for example, in cell nuclei, organelles, protoplasm, and membranes, as well as the inter-cellular space, and in body fluids such as blood. Despite their ubiquity, proteins are extremely sensitive to their environments and thus are not always easy to detect and to identify because they can degrade very quickly.
A protein is composed of one or more strings (polypeptide chains) of the residues of the 20 naturally occurring amino acids, which fold into specific 2- and 3-dimensional structures that determine the protein's activity. A given protein has a unique sequence of amino acids, termed its primary structure. The secondary structure is defined by dihedral angles (referred to as phi and psi) of the backbone atoms of the amino acid residues, and the hydrogen bonds between side chain and backbone atoms. The dihedral angles and patterns of hydrogen bonds within certain characteristic subsequences of consecutive (and non-consecutive) residues, can give rise to units of secondary structure that are relatively stable, e.g., so-called alpha helices, and beta sheets.
The tertiary structure of a protein is the term used to refer to how the secondary structure units and the polypeptide chains that connect them fold into a three dimensional structure. The quaternary structure refers to how two or more non-contiguous polypeptide chains that each adopt their own tertiary structure also associate with one another to form a protein. A protein's function may derive from either or both of its tertiary and quaternary structure. Typically the three-dimensional conformations adopted by the one or more polypeptide chains give rise to features, often described as clefts, cavities, or grooves depending on their geometry, that can bind to other molecules with high specificity. Such other molecules include drugs, nucleic acids, and most significantly for sample integrity, other proteins and polypeptides.
The natural functions of the assemblage of proteins in an organism are kept in check by a complex but delicate balance of biochemical pathways while the organism is alive. Once an organism dies, or once a sample of tissue is extracted from a living organism, the regulatory balance of the organism or in the sample is lost and key proteins start to break down. The breakdown can manifest itself in a number of different ways. For example, some proteins whose natural role is to digest other proteins (a “proteolytic” function), and whose natural levels are kept in check while an organism is alive, may go out of control after death. Thus, many proteins and key polypeptides such as coactivators, hormones, and corepressors, end up being actually digested by naturally occurring proteolytic proteins in the sample. Digestion typically involves a rupturing of the polypeptide backbone at one or more points, thereby resulting in protein or peptide fragments. Still other proteins may naturally decompose by other means, such as hydrolysis; whereas in a living organism their levels are maintained because they are continually synthesized, after death they rapidly disappear. For example, post-mortem activity of proteases and oxidative stress has been shown to play an important role on peptide and protein concentration in the brain, as well as for detecting post-translational modifications (K. Sköld et al., “A Neuroproteomoic Approach to Targeting Neuropeptides in the Brain”, Proteomics, 2, 447-454, (2002); M. Svensson et al., “Peptidomics-Based Discovery of Novel Neuropeptides”, J. Proteome Res., 2, 213-219, (2003), both of which are incorporated herein by reference.
For purposes of protein identification, however, to determine what proteins are present in a sample, it is sufficient to be able to ascertain their respective primary structures, i.e., sequences. Proteins and polypeptides have been widely investigated by methods such as two dimensional gels and mass spectrometry, but such techniques depend on having access to samples in which natural protein degradation has not advanced to a point where the concentrations of critical species have been reduced below the various measurement thresholds.
To study proteins and peptides, tissue or cell samples are usually disrupted by homogenization in certain specific buffer conditions. These buffers often contain ingredients that are supposed to cause a cessation of all protein activity, including proteins (proteases) that degrade other proteins. However, the study of tissue samples from patients or model organisms usually exposes the samples to a certain period of oxygen and nutrient depletion before homogenization and protease inactivation occurs.
Consequently, techniques have been developed in the art for attempting to preserve biological samples after extraction and prior to analysis. Examples of such techniques include tissue fixation, which typically involves immersing a sample in an aldehyde solution, and irradiating samples with microwaves (see, e.g., Theodorsson, et al., “Microwave Irradiation Increases Recovery of Neuropeptides From Brain Tissues”, Peptides, 11:1191-1197, (1990)). Use of aldehyde solutions is problematic because it doesn't arrest natural degradation of proteins (though it is somewhat effective at maintaining large-scale structure of tissues). Microwave irradiation is problematic because it is generally non-uniform, that is, some parts of the sample reach a temperature that is high enough to cause sample breakdown. (See, for example, Fricker et al., “Quantitative Neuropeptidomics of Microwave-irradiated Mouse Brain and Pituitary”, Molecular & Cellular Proteomics, 4:1391-1405, (2005).) Furthermore, microwave irradiation has formerly been applied to living (non-human) subjects as part of a sacrificial protocol and thus has yet to be established as a tool for analyzing samples that have been extracted from subjects, both human and non-human.
Accordingly, there is a need for a reliable technique for preserving the contents of tissue samples prior to analysis in a way that impedes natural degradation and that acts on a given sample reliably.
The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge as at the priority date of any of the claims.
Throughout the description and claims of the specification the word “comprise” and variations thereof, such as “comprising” and “comprises”, is not intended to exclude other additives, components, integers or steps.